Equine Embryonic Interzone and of Articular Cartilage Zones

Cells from the superficial (AACS), middle (AACM) and deep (AACD) adult articular cartilage zones and the intermediate (II) and outer (OI) interzone layers and the transient embryonic cartilage of the long bone anlagen (EC) at gestational day 40 were separately collected using laser capture microdissection and microarray analysis was performed to confirm appropriate layer selection. Microarray gene expression analysis of ACCS, ACCM, ACCD, II, OI and EC (all n=1) was carried out by Miltenyi Biotech GmbH (Miltenyi Biotec, Bergisch-Gladbach, Germany). Briefly, RNA was amplified using SuperAmp (Agilent Technologies, Paolo Alto, CA, USA), a global PCR protocol using mRNA-derived cDNA, according to the manufacturer’s protocol. Yields of cDNA were measured with a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA). The integrity of the cDNA was checked via the Agilent 2100 Bioanalyzer platform. 250 ng of each of the cDNAs was labelled with Cyanine 3-CTP (Cy-3) and hybridized overnight (17 hours, 65°C) to an Agilent Whole Horse Genome Oligo Microarrays 8x60K using Agilent’s recommended hybridization chamber and oven. Fluorescence signals of the hybridized microarrays were detected using Agilent’s Microarray Scanner System. The Agilent Feature Extraction Software was used to read out and process the microarray image files